Developmental Immunology, 1990, Vol. 1, pp. 67-75 Reprints available directly from the publisher Photocopying permitted by license only
(C) 1990 Harwood Academic Publishers GmbH Printed in the United Kingdom
Ontogeny of Rat Thymic Epithelium Defined by Monoclonal Anticytokeratin Antibodies MIODRAG (OLI(*, SUZANA JOVANOVI(,
MILIJANA VASILJEVSKI, and ALEKSANDAR DUJI(
Institute for Experimental Medicine, Military Medical Academy Belgrade, Yugoslavia
Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunoflouorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall’s corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two week.s of postnatal life.
KEYWORDS: Rat thymus, cytokeratins, monoclonal antibodies, ontogeny.
INTRODUCTION Thymic epithelial cells (TEC) form a supporting network of the thymus and also play important roles in the generation of T lymphocytes. Close contact of TEC with surrounding thymocytes is an important factor in proliferation, differentiation, education, and selection of thymocytes. Interaction with major histocompatibility complex (MHC) antigens on TEC surface as well as humoral factors that they secrete are essential events in these processes (Gelfand et al., 1980). In spite of this, very little is known about the function of particular TEC subpopulations. During the last few years, a new approach to this problem has been the generation of rnonoclonal antibodies (mAbs) specific for individual thymic components. The use of these reagents enabled *Corresponding author. Present address: Institute for Experimental Medicine, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Yugoslavia.
phenotypically distinct subcapsular/perivascular, cortical, and medullary zones. In addition, a variety of other TEC compartments, especially in the medulla, has been demonstrated (Ritter and Haynes, 1987; Kampinga et al., 1989). Cytokeratins (CK) are other important markers of the thymic epithelium. MAbs specific for CK polypeptides or CK families revealed a considerable TEC heterogeneity in humans (Laster et al., 1986), mice (Savino and Dardenne, 1988a), guinea pigs (Nicolas et al., 1986), and rats ((oli4 et al., 1988a). In our recent work ((oli4 et al., 1989), using multimarker analysis of the rat TEC by a panel of mAbs recognizing individual CK polypeptides or pairs, we identified six different TEC subsets, each characterized by a different CK map. In this work, these antibodies have been applied in order to study CK expression in the rat thymus during ontogeny. The obtained data showed their different binding patterns in fetal, neonatal, and adult thymus. dissection of the thymic epithelium into
67
M. (OLI(2 et al.
68
TABLE Characteristics and Specifities of Anti-CK mAbs MAbs
Isotype
Recognized CK polypeptides
Dilution
Manufacturer
References
RPN. 1166 (CK8)
IgG1 IgG2a IgG2a IgG2b IgG1 IgG1 IgG1 IgG1
8 1, 5-8, 10, 11, 18 18 19 acidic CK 3, 10 7 13, 16 3 13, 14 t’, 17
1:5 1:50 1:5 1:5 1:5 1:20 1:5 1:50 1:50
Amersham
Lane, 1982 Lazarides, 1982. Lane, 1982 Lane, 1982 Lane, 1982 Viac and Brochier, 1983 Tolle et al., 1985 Lazarides, 1982 Cooper et al., 1985
K.8.13 RPN. 1160 RPN. 1165 RPN. 1163 KL1 RPN. 1162 K.8.12 AE5 KS.13.1
(CK18) (CK19) (CK type I)
(CK7)
IgG IgG1
ICN Amersham Amersham Amersham
Serotec Amersham
ICN ICN ICN
1:50
aNumbered according to Moll’s classification (Moll et al., 1982). Weak immunoreactivity. qmmunoreactivity given by the manufacturer.
TABLE 2 Ontogenetic Analysis of the Expression of CK Polypeptide,s in the Rat Thymus Fetal MAbs
CK8
TEC subsets
15 d
17 d
19 d
0d
7d
14 d
6 wk
SC/PV
NP + + NP NP
NP + + NP NP
NP + + NP NP
+(p) + + +
+(p) + + +
+(p) + + +
+ + + +
+(f) NP NP
+(f) NP NP +
+(f) NP NP + + NP NP +(b) +(a) NP NP
+(c) +(c)
+(c) +(c)
+(b) +(c)
+ +(c)
+(p) +(a) +(a)
+(p)
+(p)
+
+(b) +(a)
+(c) +(b)
+(b)
+(p) +(a) +(b)
+(p) +(b) +(b)
+(p) +(c) +(b)
+(b)
+(f)
+(p)
+(p)
+(p) +(c) (f,p)
C M HC CK18
SC/PV
C M HC CK19
SC/PV
C
M HC CKI
SC/PV
M HC
NP
M HC
KL1
SC/PV
C M HC K8.12
NT
NP NP
+(a) NP
+
+(f) NP NP +(f) +(a) NP NP
+(f)
+(f)
+(c)
+(f) +(a) +(a)
+(f) +(c) +(b)
+(f) +(c) +(b)
+
+
+
+(f)
+(f)
+ +(p)
+(c)
+(c)
NT
+(f) NP NP
+(f) NP
+(f)
+(c)
NT
+(f)c NP
+(f)c NP
+(c)
+(c)C +(c)
SC/PV
C M HC
+ NP NP +(b) +(a) NP NP
NP
SC/PV
M HC KS13.1
+ + NP NP +(b) +(a) NP NP
SC/PV
C CK7
Adult
+(c)
SC--subcapsule; PV--perivascular area; C--cortex; M---medulla; HC Hassall’s corpuscles; NT---nontested; NP--nonpresent; a approx. 75-90% cells positive; b approx. 25-75% cells positive; c=approx. 10-25% cells positive; p =patches of positive cells; f--few cells positive; fp few patches on some sections positive. bperipheral layer of some HC positive. "Weak positivity.
ONTOGENY OF RAT THYMIC CYTOKERATINS
RESULTS
Ontogeny of CK Expression within Rat Thymic Epithelium The use of anti-CK mAbs (Table 1) demonstrated different expressions of particular CK polypeptides in the rat TEC during ontogeny. Table 2 summarizes their main binding patterns. CK8, which is a pan-TEC marker in adult (6week-old) animals is also a pan-TEC marker during fetal ontogeny. By analyzing four main phenotypically different zones (subcapsule/perivascular area, cortex, medulla, and Hassall’s corpuscles [HC]), it can be seen that the development of subcapsular epithelium occurs in the first two postnatal weeks. In addition, clusters of medullary TEC, identified as small, atypic HC, were first observed in neonatal thymus. The same staining patterns were seen with K 8.13 mAb. During fetal ontogeny, CK18 bound to few cells in the medulla, whereas in the postnatal period, it was also gradually expressed in the cortex (Figs. 1A and 1B). Quite different staining patterns were observed using CK19 mAb. Namely, this CK subunit was mainly a pan-TEC marker in fetal thymus (Fig. 2A) and a marker for subcapsular/ perivascular and some medullary TEC in adult thymus (Fig. 2C). Loss of CK19 in cortical TEC occurred in the first 2 weeks of postnatal life (Fig. 2B). Similar, but less strong, staining patterns were seen with CK-type mAb, recognizing nonidentified acidic CK components. KL1 mAb, detecting 3/10 CK pair in mouse thymus (Savino and Dardenne, 1988a) and probably only CK10 in the rat thymus ((oli4 et al., 1989) bound to most medullary TEC in fetal thymus (Fig. 3A) as well as to a subset of medullary TEC and HC in adult rats (Fig. 3C). However, variable patches of cortical epithelium, closer to the corticomedullary region, were KLI+ in the neonatal (Fig. 3b) and early postnatal periods. CK7 and K.8.12 mAbs showed similar immunoreactivity both in adult and neonatal tymus. However, a number of positive TEC in the medulla and subcapsular/perivascular area gradually increased after birth. AE 5 mAbs detecting CK3 was nonreactive (not shown in Table 2), whereas some weak positivity with KS 13.1 mAb was seen from day 17 of fetal life till day 7 of the postnatal period.
69
published in detail ((oli4 et al., 1989). To do this, we performed various combinations of double immunofluorescence staining with anti-CK mAbs. mutually differing in isotipe specificity. The first combination, using CK18 and 19 mAbs, showed three different staining patterns. The subset of CK18+ medullary TEC was CK19+, although single CK19+ cells were more numerous (Figs. 4A and 4B). In the cortex, distinct areas of single and double positive cells were observed. Patches of CK19 + subcapsular/perivascular TEC were CK18-. A similar staining pattern was seen using a dual combination of CK18 and KL1 mAbs (not shown). Double immunostaining with CK8 and CK19 mAbs showed that CK8 + cells were predominantly CK19 +, except for some patches in the cortex and a small subset in the medulla, which were CK8 +19(Figs. 5A and 5B). Using a combination of CK19 and KL1 mAbs, we found that cortical TEC were ma.inly double positive, whereas in the medulla, three distinct TEC subsets were identified: CK19+KLl+, CK19+KL1--, and CK19-KL1 + (Figs. 6A and 6B). Testing CK18/KL1 mAbs pooled together or CK18/CK19 mAbs as a first step as well as CK19 and KL1, respectively as a second step, we identified single CK19+ (not shown) or KLI+ cells in the medulla (Figs. 7A and
7B). Finally, to check whether CK19 + and KL1 + TEC comprised the whole medullary epithelial compartment, we performed double staining using CK19 and KL1 mAbs pooled together in combination with K8.13 mAb. A discrete TEC subset positive only with K8.13 mAbs was seen (Figs. 8A and 8B). Based on the results obtained in this study, we summarized phenotypes of TEC subsets in neonatal thymus (Table 3) and compared them with the adult ones determined previously ((oli4 et al., 1989). Table 3 and Fig. 9 show the differences in CK polypeptides between neonatal and adult thymus in all phenotypic zones. The main difference was observed in the cortex. In the medulla of the neonatal thymus, we did not identify the CK8 +10-18 +19 + subset present in adult thymus. In addition, in the neonatal thymus, the percentage of CK8 +18 +19 +10 + cells was higher, whereas the percentages of CK8+18-19-10-was smaller compared to the adult patterns.
CK-Defined TEC Subsets in Neonatal Thymus Using Double Immunofluorescence Labelling
DISCUSSION
The next step in our study was to precisely define CK phenotypes of neonatal TEC and to compare them with the adult patterns, which we previously
In this study, we applied a large panel of anti-CK mAbs and demonstrated that within rat thymic
70
M. OLI( et al.
epithelium, CK are differently expressed in fetal, neonatal, and adult thymus. Various combinations of single and double immunostaining enabled precise characterization of individual TEC subpopulations. In our previous work (Coli4 et al., 1989), we identified six different TEC subsets in adult rat thymus, each characterized by different CK polypeptide expression: four separate medullary TEC, one
medullary TEC phenotypically common with subcapsular/perivascular TEC, and one cortical TEC (see also Table 3 in this work). This study extended the previous one and throws new light on TEC ontogeny. The most marked difference between fetal and adult rat thymus was seen in the cortex. In adult animals, cells in the cortex expressed CK8 and 18, a CK pair very frequently found in simple epithelia
FIGURES 1-3. Streptavidin-biotin immunoperoxidase staining of the rat thymus with anti-CK mAbs: 1.(a) Neonatal and (b) adult thymus stained with CK18 mAb. Note a subset of positive cells in the medulla (M) in both figures. Patches of positive cells in the cortex (C) of neonatal thymus or almost all cells in adult thymus are seen, while the subcapsule (arrow) is negative. 2.(a) Fetal (17 days), (b) neonatal, and (c) adult thymus stained with CK19 mAb. Note mainly panepithelial staining in fetal thymus. Most medullary TEC (M), patches of cortical TEC (C), and a part of subcapsular/subtrabecular TEC (arrow) are positive in neonatal thymus. A subset of medullary TEC (M) and subcapsular TEC (arrow) are positive in adult thymus. Cortical TEC (C) are negative. 3.(a) Fetal (17 days), (b) neonatal, and (c) adult thymus stained with KL1 mAb. Substantial numbers of medullary TEC are stained in fetal thymus. Most medullary TEC (M) and patches of cortical TEC (C) are positive in neonatal thymus, whereas the subcapsule (arrow) is negative. A subset of medullary TEC (M) is positive in adult thymus, and no staining in the cortex (C) and subcapsule (arrow) is seen. Bar for all figures =80/zm. (See Colour Plate at the back of this publication.)
ONTOGENY OF RAT THYMIC CYTOKERATINS
(Cooper et al., 1985), whereas in fetal life, CK8 and 19 were present. Cortical TEC, both in fetal and adult mice, possess the same CK8/18 polypeptides (Savino and Dardenne, 1988a). CK19 is expressed in glandular tissues, independent of their origin and in some nonkeratinized stratified epithelia (Quinlan et al., 1985). Although this CK is mainly a pan-TEC marker during fetal ontogeny, in adult rat thymus, its expression is restricted to subcapsular/perivascular epithelium and to a subset of medullary cells. In addition, we previously demonstrated rare CK19 + cysts in the rat thymus ((oli4 et al., 1988a). Subsets of medullary TEC, both in adult-and fetal mice positive with
71
CK19, were described by Savino and Dardenne
(1988a). Interestingly, during the first two postnatal weeks in rats, simultaneous loss of CK19 and the expression of CK19 occurred in the cortex. We did not find similar data in the literature, but partial or complete loss of the epitopes recognized by the anti-CK19 mAb was detected in some thymoma (Cooper et al., 1985; Savino and Dardenne, 1988b). The epitopes are probably masked in situ and cannot be detected in tissue sections, but immunoblot analysis revealed their presence under denaturing conditions (Cooper et al., 1985; Savino and Dardenne, 1988b). We found that reexpression of CK19 in a patchy pattern in
FIGURES 4-6. Double immunofluorescence staining of neonatal rat thymus with anti-CK mAbs: 4.(a) CK19 visualized with TR, and (b) CK18 visualized with FITC. In the medulla, a cluster of double positive cells is indicated by large arrows. In addition, more single positive cells (small circle) is present. In the cortex, patches of CK18 +19 weakly +cells (small arrows) and single CK19 + cells are seen. 5.(a) CK19 visualized with TR, and (b) CK8 visualized with FITC. Note the double positive and single CK8 + cells (arrows) in the medulla. 6.(a) CK19 visualized with TR, and (b) KL1 visualized with F!TC. Some double positive cells (double arrows), single CK19+ cells (large arrows), and single KL1 + cells (astericks) in the medulla are indicated. Bar for all figures 20/m. (See Colour Plate II at the back of this publication.)
M.
72
COLIC
et al.
FIGURES 7 and 8. Double immunofluorescence staining of neonatal thymus with anti-CK mAbs: 7.(a) CK18 and 19 mAbs pooled together and visualized with TR, and (b) KL1 mAb visualized with FITC. Single KL1 + cells in the medulla are arrowed. 8.(a) CK19 and KL1 mAbs pooled together and visualized with TR, and (b) K.8.13 mAb visualized with FITC. Single K.8.13 + cells in the medulla are arrowed. Bar for all figures- 20/m. (See Colour Plate III at the back of this publication.)
A
B SUB CAPSUL E-
CORTEX
MEDULLA
+
+
CK 8 10 18-19-
CK 6’/ 10- 18" 19 + FIGURE 9.
Shematic representation of CK-defined TEC subsets in (A) neonatal and (B) adult rat thymus.
ONTOGENY OF RAT THYMIC CYTOKERATINS
outer thymic cortex of adult rats recovering after sublethal X-ray irradiation (nonpublished observations). It is not clear what is the significance of this phenomenon. TABLE 3 Cytokeratin-Defined TEC Subsets in Neonatal and Adult Rat Thymus Localization
TEC subsets Neonatal thymus
Adult thymus
CK8 +10 -18 -19 +b
CK8 +10 -18 -19 +
Cortex
CK8 +10 +18 -19 + CK8 +10-18 +19CK8 +10 +18 +19 +
CK8 +10 -18 +19
Medulla
CK8 +10 +18 +19 + CK8 +10-18-19 + CK8 +10 +18-19CK8 +10 -18-19-
CK8 +10 +18 +19 + CK8 +10-18-19 + CK8 +10 +18-19CK8 +10-18-19CK8 +10 -18 +19 +
Subcapsule/ perivascular.area
aTEC subsets in adult thymus are determined previously ((oli4 et al., 1989). bOnly patches of TEC are positive (staining with CK10 in this subset is given as negative although some rare cells express CK10 polypeptide).
More complex CK organization was found in the medulla, both in neonatal and adult thymus. Most medullary TEC share common CK polypeptides either with subcapsular/perivascular TEC or with cortical TEC. CK8/KLI+ cells or cells positive only with CK8 are two separate TEC subsets found only in the medulla. KL1 mAb recognizing 3/10 CK pair in mice (Savino and Dardenne, 1988a) probably detects CK10 in rats, because we did not identify any positive cells with AE5 mAb specific for CK3. KL1 recognizes only CK10 in human thymus, too (Haftek et al., 1986). The positivity of HC and a subset medullary TEC with KL1 mAb in adult rat ((oli4 et al., 1989; this work) and mouse (Nicolas et al., 1985) or HC in guinea pig (Nicolas et al., 1986) indicates that CK10 is a marker for terminally differentiated thymic epithelium. Our data presented here clearly show that this CK subunit has a different distribution during ontogeny. Namely, in the early postnatal period, CK10 is expressed in most medullary TEC and also in a patchy pattern in the cortex, predominantly together with CK19 polypeptide. However, single CK10+ cells were present as early as 17 days of fetal life (not shown) and their numbers increased with age. It is not clear whether these cells originate from a common CK10+19+ subpopulation or not.
73
Subcapsular/perivascular TEC share common CK polypeptides 7, 8, 16, and 19 with a subset of medullary TEC, indicating their common origin. A panel of monoclonal antibodies with the same specificity has been produced in the human thymus (reviewed by Ritter and Haynes, 1987) or in the rat thymus ((oli et al., 1988b; Kampinga et al., 1989). Our data showed that the subcapsular epithelium in the rat thymus started to develop after birth. The patches of positive cells were first seen in a deep paraseptal area, sometimes close to the central medullary zone. The appearance of this cell layer in fetal human thymus was seen earlier, at 12 weeks of gestation (Lobach and Haynes, 1987). Our results are in accordance with those published by Crouse et al. (1985). They described a mechanism in which an outgrowth of epithelial cells from a central core in the thymic primordium (branchial cleft) surrounds the outer branchial pouch and forms the subcapsular zone. The main question resulting from this study is the interrelationship of the rat TEC in terms of its origin. It is largely accepted that in mammals, thymic epithelium is derived from both endoderm (third pharyngeal pouche) and ectoderm (third pharyngeal cleft) (reviewed by Lampert and Ritter, 1988). Close physical contact between them and probably their mutual inductive signals are necessary for normal thymic development. However, 4ontroversy over the exact contribution of each compartment in development of TEC compartments has been described. First studies indicated that medullary epithelium, due to its central localization and the persistence in nude mice mainly in forms of tubules and cysts, was endodermal, wh(reas the cortex was ectodermal in origin (Cordier and Heremans, 1975). Recent comparative investigations using a large panel of antiTEC mAbs showed that subcapsular and medullary epithelium shared many molecules with epidermal cells, indicating common ectodermal origin of the skin and thyrnic medulla (Ritter and Haynes, 1987). More recently, Lampert and Ritter (1988) suggested_ that thymic epithelium could be derived from a common epithelial stem cell. The main findings supporting this hypothesis are thymic tumors (Willcox et al., 1987) that simultaneously express both cortical and medullary TEC markers. Ontogenetic studies in mice also favor this hypothesis
(Lampert and Ritter, 1988). If we compare our ontogenetic study on CK expression in the cortex and medulla of the rat
74
M. (OLI( et al.
thymus, we cannot find any strong evidence of their and reagents such as goat antimouse IgG subclass dual origins. CK18 is the best candidate as a marker specific biotinylated antibodies and streptavidin for endodermaly derived thymic epithelium. Our coupled with perioxidase or Texas red (TR) were recent work (Mitrovic et al., 1989) showed that in purchased from Amersham International, United rats, this CK subunit was present in the simple Kingdom. Sheep antimouse IgG subclass specific epithelia, predominantly endodermal in origin. No antibodies conjugated with fluorescein isothiocynate epithelia originating from the ectoderm were posi- (FITC) were obtained from Serotec, United tire. However CK18 is not the only marker for Kingdom. cortical TEC. In addition, its distinct ontogenetic expression and the presence of adult medullary CK types" (CK10 and 19) in the cortex during the fetal Immunohistochemistry, and early postnatal periods are not in accordance For immunohistochemistry, two methods were used: with this hypothesis. Small subsets of medullary streptavidin-biotin immunoperoxidase staining and TEC and subcapsular/perivascular TEC that have double immunofluorescence staining. CK7 and 16 in all ontogenetic period as well as Streptavidin-biotin immunoperoxidase staining medullary CK18 negative cells could be the candi- was performed as follows: thymic cryostat sections dates for ectodermaly derived epithelium. But the (5/m), which were fixed in acetone for 10 min, problem is how to explain the origins of other cell were first incubated with mAbs for a minimum of types. The simultaneous expression of various 60 min, washed in TBS, pH 7.6 for 10 min, followed common CK polypeptides during ontogeny favors, by incubation with secondary biotinylated antibodies although does not strongly prove, the hypothesis of (IgG subclass specific) diluted 1:100 in TBS for a common origin of thymic epithelium. Medullary 30 min. After washing in TBS, sections were incucells having common and constant CK polypeptides bated with streptavidin-peroxidase (1:100) for 8, 10, 18, and 19 in all ontogenetic periods could be 30 min. Revelation of the peroxidase activity was a common, maybe undifferentiated, TEC subset, performed by a 10-min incubation of the sections in Morphologically undifferentiated epithelial cells have 0.06% DAB (Serva, FRG) in 0.01% H202. Finally, been previously described in the corticomedullary slides were lightly counterstained with hematoxylin region and medulla (yon Gaudecher et al., 1986). and mounted in gelatin/glycerol medium. In conclusion, this work throws new light on the Double immunofluorescence was performed in development of thymic epithelium and raises many order to determine whether two different CK polyquestions about the functional significance of so peptides were present in the, same TEC. To prove extremely different CK polypeptide expressions in this, sections were incubated with mAbs mutually the thymus, differing in isotype specificity. The reaction was visualized using subclass-specific secondary reagents coupled with different dyes. The staining sequence MATERIALS AND METHODS was as follows: first, mAb, IgG subclass-specific FITC-coupled antibody; second, mAb .IgG subclass Animals specific biotinylated antibody, streptavidin-TR. Thymuses were obtained from A0 rats, bred at the Sometimes an opposite sequence of secondary antiFarm for Experimental Animals, Military" Medical bodies was applied. After washing following each Academy, Belgrade, of the following age groups: incubation, sections were mounted in glycerol and days 15, 17, and 19 of fetal life, day of birth, days 7 observed for green and red fluorescence under a and 14 or 6 weeks of postnatal life. Day 0 of preg- fluorescence microscope (Univar III). Specificity of nancy was determined by regular controls of the labeling was checked using appropriate controls, estral cycle and findings of spermatozoides in including single staining, omitting the first or vaginal smears. Samples were collected from 3-5 second mAb, as well as their replacement with an animals per time point, irrelevant mouse mAb of the same isotype (produced in our laboratory). All controls gave negative Antibodies and Reagents
results.
A panel of 10 mouse, anti-CK monoclonal antibodies
(Received August 20, 1989)
was used. Their specificity, isotype, dilution, and origin were given in Table 1. Secondary antibodies
(Accepted September 26, 1989)
ONTOGENY OF RAT THYMIC CYTOKERATINS
REFERENCES (oli4 M., Matanovi4 D., Hegadi Lj., and Duji4 A. (1988a). Heterogeneity of rat thymic epithelium by monoclonal antikeratin antibodies. Thymus 12: 123. (oli4 M., Matanovi4 D., Hegedi Lj., and Duji4 A. (1988b). Immunohistochemical characterization of rat thymic nonlymphoid cellsmI. Epithelial and mesenchymal components defined by monoclonal antibodies. Immunology 65: 277. (oli4. M., Jovanovi4 S., Mitrovi4 S., and Duji4 A. (1989). Immunohistochemical identification of six cytokeratin-defined subsets of the rat thymic epithelial cells. Thymus 13: 175. Cooper D., Schermer A., and Sun T.T. (1985). Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: Strategies, applications, and limitations. Lab. Invest. 52: 243. Cordier A.C., and Heremans J.E. (1975). Nude mouse embryo: Ectodermal nature of the primordial thymic defect. Scand. J. Immunol. 4: 193. Crouse D.A., Turpen J.B., and Sharp J.G. (1985). Thymic nonlymphoid cells. Surv. Immunol. Res. 4: 120. Gaudecker B. Von. (1986). The development of the human thymus microenvironments. In: The human thymus: Histophysiology and pathology, Muller-Hermelink H.K., Ed. (Berlin: Springer
Verlag), p. 1. Gelfand E.W., Dosch H.M., Shore A., Limbatibul S., and Lee J.W.W. (1980). Role of thymus in human T cells differentiation. In: Biological basis of immunodeficiency, Gelfand E.W., and Dosch H.M., Eds. (New York: Raven Press), p. 39. Haftek M., Staquet M.J., Viac J., Schmitt D., and Thivolet J. (1986). Immunogold labeling of keratin filaments in normal human epidermal cells with two anti-keratin monoclonal antibodies. J. Histochem. Cytochem. 34: 613. Kampinga J., Berger S., Boyd R., Brekelmans P., Coli4 M., Van Ewijk W., Kendall M., Ladyman H., Nieuwenhuis P., Ritter M., Schuurman H-J., and Tournefier A. (1989). Thymic epithelial antibodies: Immunohistological analysis and introduction of CTES nomenclature. Thymus 13: 165. Lampert I., and Ritter M.A. (1988). The origin of the diverse epithelial cells of the thymus: Is there a common stem cell? In: Thymus update, vol. 1, Kendall M.D., and Ritter M.A., eds. (London: Harwood Academic Publishers), p. 5. Lane E.B. (1982). Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J. Cell Biol. 92: 665. Laster A.J., Itoh T., Palker T.J., and Haynes B.F. (1986). The human thymic microenvironment: Thymic epithelium contain specific keratins associated with early and late stages of epidermal keratinocyte maturation. Differentiation. 31: 67.
75
Lazarides E. (1982). Intermediate filaments: A chemically heterogeneous, developmentally regulated class of proteins. Annu.. Rev. Biochem. 51: 219. Lobach D.F., and .Haynes B.F. (1987). Ontogeny of the human thymus during fetal development. J. Clin. Immunol. 7: 81. Mitrovi4 S., (oli4 M., and Popovi4 Lj. (in press). Comparative studies on the expression of SHC-9D8 antigen and cytokeratin 18 in rat epithelial cells. Period. Biol. Moll R., Franke W.W., and Schiller D.L. (1982). The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell 31: 11. Nicolas J.F., Reano A., Kaiserlian D., and Thivolet J. (1986). Epithelial cell heterogeneity in the guinea pig thymus: Immunohistochemical characterization of four thymic epithelial subsets defined by monoclonal antikeratin antibodies. Eur. J. Immunol. 16: 457. Nicolas J.F., Savino W., Reano A., Viac J., and Dardenne M. (1985). Heterogeneity of thymic epithelial cell (TEC) keratins: Immunohistochemical and biochemical evidence for a subset of highly differentiated TEC in the mouse. J. Histochem. Cytochem. 33: 687. Quinlan R.A., Schiller D.L., Hatzfeld M., Achstatter T., Moll R., Jorcano J.L., Magin T.M., and Franke W.W. (1985). Patterns of expression and organization of cytokeratin intermediate filaments. Annu. NY Acad. Sci. 455: 282. Ritter M.A., and Haynes B.F. (1987). Summary of thymic epithelium workshop. In: Leucocyte typing III. White cell differentiation antigens, McMichael, A., Beverley P., Hagg N., and Horton M. Eds. (Oxford: Oxford University Press), p. 247. Savino W., and Dardenne M. (1988a). Development studies on expression of monoclonal antibody-defined cytokeratins by thymic epithelial cells from normal and autoimmune mice. J. Histochem. Cytochem. 36: 1123. Savino W., and Dardenne M. (1988b). Immunohistochemical studies on a human thymic epithelial subset defined by the anti-cytokeratin 18 monoclonal antibody. Cell Tissue Res. 254: 255. Tolle H.G., Weber K., and Osborn M. (1985). Microinjection of monoclonal antibodies specific one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments. Eur. J. Cell Biol. 38: 234. Viac J., Reano A., Brochier J., Staquet M.J., Thivolet J. (1983). Reactivity pattern of monoclonal anti-keratin antibody.(KL 1). J. Invest. Dermatol. 81: 351. Willcox N., Shluep M., Ritter M.A., Schuurman H-J., NewsonDavis J., and Christensson B. (1987). Myasthenic and nonmyasthenic thymoma. An expansion of a minor cortical epithelial cell subset? Am. J. Pathol. 127: 447.
MEDIATORS of
INFLAMMATION
The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Gastroenterology Research and Practice Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Journal of
Hindawi Publishing Corporation http://www.hindawi.com
Diabetes Research Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
International Journal of
Journal of
Endocrinology
Immunology Research Hindawi Publishing Corporation http://www.hindawi.com
Disease Markers
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Volume 2014
Submit your manuscripts at http://www.hindawi.com BioMed Research International
PPAR Research Hindawi Publishing Corporation http://www.hindawi.com
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Volume 2014
Journal of
Obesity
Journal of
Ophthalmology Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Evidence-Based Complementary and Alternative Medicine
Stem Cells International Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Journal of
Oncology Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Parkinson’s Disease
Computational and Mathematical Methods in Medicine Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
AIDS
Behavioural Neurology Hindawi Publishing Corporation http://www.hindawi.com
Research and Treatment Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Oxidative Medicine and Cellular Longevity Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
(C) 1990 Harwood Academic Publishers GmbH Printed in the United Kingdom
Ontogeny of Rat Thymic Epithelium Defined by Monoclonal Anticytokeratin Antibodies MIODRAG (OLI(*, SUZANA JOVANOVI(,
MILIJANA VASILJEVSKI, and ALEKSANDAR DUJI(
Institute for Experimental Medicine, Military Medical Academy Belgrade, Yugoslavia
Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunoflouorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall’s corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two week.s of postnatal life.
KEYWORDS: Rat thymus, cytokeratins, monoclonal antibodies, ontogeny.
INTRODUCTION Thymic epithelial cells (TEC) form a supporting network of the thymus and also play important roles in the generation of T lymphocytes. Close contact of TEC with surrounding thymocytes is an important factor in proliferation, differentiation, education, and selection of thymocytes. Interaction with major histocompatibility complex (MHC) antigens on TEC surface as well as humoral factors that they secrete are essential events in these processes (Gelfand et al., 1980). In spite of this, very little is known about the function of particular TEC subpopulations. During the last few years, a new approach to this problem has been the generation of rnonoclonal antibodies (mAbs) specific for individual thymic components. The use of these reagents enabled *Corresponding author. Present address: Institute for Experimental Medicine, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Yugoslavia.
phenotypically distinct subcapsular/perivascular, cortical, and medullary zones. In addition, a variety of other TEC compartments, especially in the medulla, has been demonstrated (Ritter and Haynes, 1987; Kampinga et al., 1989). Cytokeratins (CK) are other important markers of the thymic epithelium. MAbs specific for CK polypeptides or CK families revealed a considerable TEC heterogeneity in humans (Laster et al., 1986), mice (Savino and Dardenne, 1988a), guinea pigs (Nicolas et al., 1986), and rats ((oli4 et al., 1988a). In our recent work ((oli4 et al., 1989), using multimarker analysis of the rat TEC by a panel of mAbs recognizing individual CK polypeptides or pairs, we identified six different TEC subsets, each characterized by a different CK map. In this work, these antibodies have been applied in order to study CK expression in the rat thymus during ontogeny. The obtained data showed their different binding patterns in fetal, neonatal, and adult thymus. dissection of the thymic epithelium into
67
M. (OLI(2 et al.
68
TABLE Characteristics and Specifities of Anti-CK mAbs MAbs
Isotype
Recognized CK polypeptides
Dilution
Manufacturer
References
RPN. 1166 (CK8)
IgG1 IgG2a IgG2a IgG2b IgG1 IgG1 IgG1 IgG1
8 1, 5-8, 10, 11, 18 18 19 acidic CK 3, 10 7 13, 16 3 13, 14 t’, 17
1:5 1:50 1:5 1:5 1:5 1:20 1:5 1:50 1:50
Amersham
Lane, 1982 Lazarides, 1982. Lane, 1982 Lane, 1982 Lane, 1982 Viac and Brochier, 1983 Tolle et al., 1985 Lazarides, 1982 Cooper et al., 1985
K.8.13 RPN. 1160 RPN. 1165 RPN. 1163 KL1 RPN. 1162 K.8.12 AE5 KS.13.1
(CK18) (CK19) (CK type I)
(CK7)
IgG IgG1
ICN Amersham Amersham Amersham
Serotec Amersham
ICN ICN ICN
1:50
aNumbered according to Moll’s classification (Moll et al., 1982). Weak immunoreactivity. qmmunoreactivity given by the manufacturer.
TABLE 2 Ontogenetic Analysis of the Expression of CK Polypeptide,s in the Rat Thymus Fetal MAbs
CK8
TEC subsets
15 d
17 d
19 d
0d
7d
14 d
6 wk
SC/PV
NP + + NP NP
NP + + NP NP
NP + + NP NP
+(p) + + +
+(p) + + +
+(p) + + +
+ + + +
+(f) NP NP
+(f) NP NP +
+(f) NP NP + + NP NP +(b) +(a) NP NP
+(c) +(c)
+(c) +(c)
+(b) +(c)
+ +(c)
+(p) +(a) +(a)
+(p)
+(p)
+
+(b) +(a)
+(c) +(b)
+(b)
+(p) +(a) +(b)
+(p) +(b) +(b)
+(p) +(c) +(b)
+(b)
+(f)
+(p)
+(p)
+(p) +(c) (f,p)
C M HC CK18
SC/PV
C M HC CK19
SC/PV
C
M HC CKI
SC/PV
M HC
NP
M HC
KL1
SC/PV
C M HC K8.12
NT
NP NP
+(a) NP
+
+(f) NP NP +(f) +(a) NP NP
+(f)
+(f)
+(c)
+(f) +(a) +(a)
+(f) +(c) +(b)
+(f) +(c) +(b)
+
+
+
+(f)
+(f)
+ +(p)
+(c)
+(c)
NT
+(f) NP NP
+(f) NP
+(f)
+(c)
NT
+(f)c NP
+(f)c NP
+(c)
+(c)C +(c)
SC/PV
C M HC
+ NP NP +(b) +(a) NP NP
NP
SC/PV
M HC KS13.1
+ + NP NP +(b) +(a) NP NP
SC/PV
C CK7
Adult
+(c)
SC--subcapsule; PV--perivascular area; C--cortex; M---medulla; HC Hassall’s corpuscles; NT---nontested; NP--nonpresent; a approx. 75-90% cells positive; b approx. 25-75% cells positive; c=approx. 10-25% cells positive; p =patches of positive cells; f--few cells positive; fp few patches on some sections positive. bperipheral layer of some HC positive. "Weak positivity.
ONTOGENY OF RAT THYMIC CYTOKERATINS
RESULTS
Ontogeny of CK Expression within Rat Thymic Epithelium The use of anti-CK mAbs (Table 1) demonstrated different expressions of particular CK polypeptides in the rat TEC during ontogeny. Table 2 summarizes their main binding patterns. CK8, which is a pan-TEC marker in adult (6week-old) animals is also a pan-TEC marker during fetal ontogeny. By analyzing four main phenotypically different zones (subcapsule/perivascular area, cortex, medulla, and Hassall’s corpuscles [HC]), it can be seen that the development of subcapsular epithelium occurs in the first two postnatal weeks. In addition, clusters of medullary TEC, identified as small, atypic HC, were first observed in neonatal thymus. The same staining patterns were seen with K 8.13 mAb. During fetal ontogeny, CK18 bound to few cells in the medulla, whereas in the postnatal period, it was also gradually expressed in the cortex (Figs. 1A and 1B). Quite different staining patterns were observed using CK19 mAb. Namely, this CK subunit was mainly a pan-TEC marker in fetal thymus (Fig. 2A) and a marker for subcapsular/ perivascular and some medullary TEC in adult thymus (Fig. 2C). Loss of CK19 in cortical TEC occurred in the first 2 weeks of postnatal life (Fig. 2B). Similar, but less strong, staining patterns were seen with CK-type mAb, recognizing nonidentified acidic CK components. KL1 mAb, detecting 3/10 CK pair in mouse thymus (Savino and Dardenne, 1988a) and probably only CK10 in the rat thymus ((oli4 et al., 1989) bound to most medullary TEC in fetal thymus (Fig. 3A) as well as to a subset of medullary TEC and HC in adult rats (Fig. 3C). However, variable patches of cortical epithelium, closer to the corticomedullary region, were KLI+ in the neonatal (Fig. 3b) and early postnatal periods. CK7 and K.8.12 mAbs showed similar immunoreactivity both in adult and neonatal tymus. However, a number of positive TEC in the medulla and subcapsular/perivascular area gradually increased after birth. AE 5 mAbs detecting CK3 was nonreactive (not shown in Table 2), whereas some weak positivity with KS 13.1 mAb was seen from day 17 of fetal life till day 7 of the postnatal period.
69
published in detail ((oli4 et al., 1989). To do this, we performed various combinations of double immunofluorescence staining with anti-CK mAbs. mutually differing in isotipe specificity. The first combination, using CK18 and 19 mAbs, showed three different staining patterns. The subset of CK18+ medullary TEC was CK19+, although single CK19+ cells were more numerous (Figs. 4A and 4B). In the cortex, distinct areas of single and double positive cells were observed. Patches of CK19 + subcapsular/perivascular TEC were CK18-. A similar staining pattern was seen using a dual combination of CK18 and KL1 mAbs (not shown). Double immunostaining with CK8 and CK19 mAbs showed that CK8 + cells were predominantly CK19 +, except for some patches in the cortex and a small subset in the medulla, which were CK8 +19(Figs. 5A and 5B). Using a combination of CK19 and KL1 mAbs, we found that cortical TEC were ma.inly double positive, whereas in the medulla, three distinct TEC subsets were identified: CK19+KLl+, CK19+KL1--, and CK19-KL1 + (Figs. 6A and 6B). Testing CK18/KL1 mAbs pooled together or CK18/CK19 mAbs as a first step as well as CK19 and KL1, respectively as a second step, we identified single CK19+ (not shown) or KLI+ cells in the medulla (Figs. 7A and
7B). Finally, to check whether CK19 + and KL1 + TEC comprised the whole medullary epithelial compartment, we performed double staining using CK19 and KL1 mAbs pooled together in combination with K8.13 mAb. A discrete TEC subset positive only with K8.13 mAbs was seen (Figs. 8A and 8B). Based on the results obtained in this study, we summarized phenotypes of TEC subsets in neonatal thymus (Table 3) and compared them with the adult ones determined previously ((oli4 et al., 1989). Table 3 and Fig. 9 show the differences in CK polypeptides between neonatal and adult thymus in all phenotypic zones. The main difference was observed in the cortex. In the medulla of the neonatal thymus, we did not identify the CK8 +10-18 +19 + subset present in adult thymus. In addition, in the neonatal thymus, the percentage of CK8 +18 +19 +10 + cells was higher, whereas the percentages of CK8+18-19-10-was smaller compared to the adult patterns.
CK-Defined TEC Subsets in Neonatal Thymus Using Double Immunofluorescence Labelling
DISCUSSION
The next step in our study was to precisely define CK phenotypes of neonatal TEC and to compare them with the adult patterns, which we previously
In this study, we applied a large panel of anti-CK mAbs and demonstrated that within rat thymic
70
M. OLI( et al.
epithelium, CK are differently expressed in fetal, neonatal, and adult thymus. Various combinations of single and double immunostaining enabled precise characterization of individual TEC subpopulations. In our previous work (Coli4 et al., 1989), we identified six different TEC subsets in adult rat thymus, each characterized by different CK polypeptide expression: four separate medullary TEC, one
medullary TEC phenotypically common with subcapsular/perivascular TEC, and one cortical TEC (see also Table 3 in this work). This study extended the previous one and throws new light on TEC ontogeny. The most marked difference between fetal and adult rat thymus was seen in the cortex. In adult animals, cells in the cortex expressed CK8 and 18, a CK pair very frequently found in simple epithelia
FIGURES 1-3. Streptavidin-biotin immunoperoxidase staining of the rat thymus with anti-CK mAbs: 1.(a) Neonatal and (b) adult thymus stained with CK18 mAb. Note a subset of positive cells in the medulla (M) in both figures. Patches of positive cells in the cortex (C) of neonatal thymus or almost all cells in adult thymus are seen, while the subcapsule (arrow) is negative. 2.(a) Fetal (17 days), (b) neonatal, and (c) adult thymus stained with CK19 mAb. Note mainly panepithelial staining in fetal thymus. Most medullary TEC (M), patches of cortical TEC (C), and a part of subcapsular/subtrabecular TEC (arrow) are positive in neonatal thymus. A subset of medullary TEC (M) and subcapsular TEC (arrow) are positive in adult thymus. Cortical TEC (C) are negative. 3.(a) Fetal (17 days), (b) neonatal, and (c) adult thymus stained with KL1 mAb. Substantial numbers of medullary TEC are stained in fetal thymus. Most medullary TEC (M) and patches of cortical TEC (C) are positive in neonatal thymus, whereas the subcapsule (arrow) is negative. A subset of medullary TEC (M) is positive in adult thymus, and no staining in the cortex (C) and subcapsule (arrow) is seen. Bar for all figures =80/zm. (See Colour Plate at the back of this publication.)
ONTOGENY OF RAT THYMIC CYTOKERATINS
(Cooper et al., 1985), whereas in fetal life, CK8 and 19 were present. Cortical TEC, both in fetal and adult mice, possess the same CK8/18 polypeptides (Savino and Dardenne, 1988a). CK19 is expressed in glandular tissues, independent of their origin and in some nonkeratinized stratified epithelia (Quinlan et al., 1985). Although this CK is mainly a pan-TEC marker during fetal ontogeny, in adult rat thymus, its expression is restricted to subcapsular/perivascular epithelium and to a subset of medullary cells. In addition, we previously demonstrated rare CK19 + cysts in the rat thymus ((oli4 et al., 1988a). Subsets of medullary TEC, both in adult-and fetal mice positive with
71
CK19, were described by Savino and Dardenne
(1988a). Interestingly, during the first two postnatal weeks in rats, simultaneous loss of CK19 and the expression of CK19 occurred in the cortex. We did not find similar data in the literature, but partial or complete loss of the epitopes recognized by the anti-CK19 mAb was detected in some thymoma (Cooper et al., 1985; Savino and Dardenne, 1988b). The epitopes are probably masked in situ and cannot be detected in tissue sections, but immunoblot analysis revealed their presence under denaturing conditions (Cooper et al., 1985; Savino and Dardenne, 1988b). We found that reexpression of CK19 in a patchy pattern in
FIGURES 4-6. Double immunofluorescence staining of neonatal rat thymus with anti-CK mAbs: 4.(a) CK19 visualized with TR, and (b) CK18 visualized with FITC. In the medulla, a cluster of double positive cells is indicated by large arrows. In addition, more single positive cells (small circle) is present. In the cortex, patches of CK18 +19 weakly +cells (small arrows) and single CK19 + cells are seen. 5.(a) CK19 visualized with TR, and (b) CK8 visualized with FITC. Note the double positive and single CK8 + cells (arrows) in the medulla. 6.(a) CK19 visualized with TR, and (b) KL1 visualized with F!TC. Some double positive cells (double arrows), single CK19+ cells (large arrows), and single KL1 + cells (astericks) in the medulla are indicated. Bar for all figures 20/m. (See Colour Plate II at the back of this publication.)
M.
72
COLIC
et al.
FIGURES 7 and 8. Double immunofluorescence staining of neonatal thymus with anti-CK mAbs: 7.(a) CK18 and 19 mAbs pooled together and visualized with TR, and (b) KL1 mAb visualized with FITC. Single KL1 + cells in the medulla are arrowed. 8.(a) CK19 and KL1 mAbs pooled together and visualized with TR, and (b) K.8.13 mAb visualized with FITC. Single K.8.13 + cells in the medulla are arrowed. Bar for all figures- 20/m. (See Colour Plate III at the back of this publication.)
A
B SUB CAPSUL E-
CORTEX
MEDULLA
+
+
CK 8 10 18-19-
CK 6’/ 10- 18" 19 + FIGURE 9.
Shematic representation of CK-defined TEC subsets in (A) neonatal and (B) adult rat thymus.
ONTOGENY OF RAT THYMIC CYTOKERATINS
outer thymic cortex of adult rats recovering after sublethal X-ray irradiation (nonpublished observations). It is not clear what is the significance of this phenomenon. TABLE 3 Cytokeratin-Defined TEC Subsets in Neonatal and Adult Rat Thymus Localization
TEC subsets Neonatal thymus
Adult thymus
CK8 +10 -18 -19 +b
CK8 +10 -18 -19 +
Cortex
CK8 +10 +18 -19 + CK8 +10-18 +19CK8 +10 +18 +19 +
CK8 +10 -18 +19
Medulla
CK8 +10 +18 +19 + CK8 +10-18-19 + CK8 +10 +18-19CK8 +10 -18-19-
CK8 +10 +18 +19 + CK8 +10-18-19 + CK8 +10 +18-19CK8 +10-18-19CK8 +10 -18 +19 +
Subcapsule/ perivascular.area
aTEC subsets in adult thymus are determined previously ((oli4 et al., 1989). bOnly patches of TEC are positive (staining with CK10 in this subset is given as negative although some rare cells express CK10 polypeptide).
More complex CK organization was found in the medulla, both in neonatal and adult thymus. Most medullary TEC share common CK polypeptides either with subcapsular/perivascular TEC or with cortical TEC. CK8/KLI+ cells or cells positive only with CK8 are two separate TEC subsets found only in the medulla. KL1 mAb recognizing 3/10 CK pair in mice (Savino and Dardenne, 1988a) probably detects CK10 in rats, because we did not identify any positive cells with AE5 mAb specific for CK3. KL1 recognizes only CK10 in human thymus, too (Haftek et al., 1986). The positivity of HC and a subset medullary TEC with KL1 mAb in adult rat ((oli4 et al., 1989; this work) and mouse (Nicolas et al., 1985) or HC in guinea pig (Nicolas et al., 1986) indicates that CK10 is a marker for terminally differentiated thymic epithelium. Our data presented here clearly show that this CK subunit has a different distribution during ontogeny. Namely, in the early postnatal period, CK10 is expressed in most medullary TEC and also in a patchy pattern in the cortex, predominantly together with CK19 polypeptide. However, single CK10+ cells were present as early as 17 days of fetal life (not shown) and their numbers increased with age. It is not clear whether these cells originate from a common CK10+19+ subpopulation or not.
73
Subcapsular/perivascular TEC share common CK polypeptides 7, 8, 16, and 19 with a subset of medullary TEC, indicating their common origin. A panel of monoclonal antibodies with the same specificity has been produced in the human thymus (reviewed by Ritter and Haynes, 1987) or in the rat thymus ((oli et al., 1988b; Kampinga et al., 1989). Our data showed that the subcapsular epithelium in the rat thymus started to develop after birth. The patches of positive cells were first seen in a deep paraseptal area, sometimes close to the central medullary zone. The appearance of this cell layer in fetal human thymus was seen earlier, at 12 weeks of gestation (Lobach and Haynes, 1987). Our results are in accordance with those published by Crouse et al. (1985). They described a mechanism in which an outgrowth of epithelial cells from a central core in the thymic primordium (branchial cleft) surrounds the outer branchial pouch and forms the subcapsular zone. The main question resulting from this study is the interrelationship of the rat TEC in terms of its origin. It is largely accepted that in mammals, thymic epithelium is derived from both endoderm (third pharyngeal pouche) and ectoderm (third pharyngeal cleft) (reviewed by Lampert and Ritter, 1988). Close physical contact between them and probably their mutual inductive signals are necessary for normal thymic development. However, 4ontroversy over the exact contribution of each compartment in development of TEC compartments has been described. First studies indicated that medullary epithelium, due to its central localization and the persistence in nude mice mainly in forms of tubules and cysts, was endodermal, wh(reas the cortex was ectodermal in origin (Cordier and Heremans, 1975). Recent comparative investigations using a large panel of antiTEC mAbs showed that subcapsular and medullary epithelium shared many molecules with epidermal cells, indicating common ectodermal origin of the skin and thyrnic medulla (Ritter and Haynes, 1987). More recently, Lampert and Ritter (1988) suggested_ that thymic epithelium could be derived from a common epithelial stem cell. The main findings supporting this hypothesis are thymic tumors (Willcox et al., 1987) that simultaneously express both cortical and medullary TEC markers. Ontogenetic studies in mice also favor this hypothesis
(Lampert and Ritter, 1988). If we compare our ontogenetic study on CK expression in the cortex and medulla of the rat
74
M. (OLI( et al.
thymus, we cannot find any strong evidence of their and reagents such as goat antimouse IgG subclass dual origins. CK18 is the best candidate as a marker specific biotinylated antibodies and streptavidin for endodermaly derived thymic epithelium. Our coupled with perioxidase or Texas red (TR) were recent work (Mitrovic et al., 1989) showed that in purchased from Amersham International, United rats, this CK subunit was present in the simple Kingdom. Sheep antimouse IgG subclass specific epithelia, predominantly endodermal in origin. No antibodies conjugated with fluorescein isothiocynate epithelia originating from the ectoderm were posi- (FITC) were obtained from Serotec, United tire. However CK18 is not the only marker for Kingdom. cortical TEC. In addition, its distinct ontogenetic expression and the presence of adult medullary CK types" (CK10 and 19) in the cortex during the fetal Immunohistochemistry, and early postnatal periods are not in accordance For immunohistochemistry, two methods were used: with this hypothesis. Small subsets of medullary streptavidin-biotin immunoperoxidase staining and TEC and subcapsular/perivascular TEC that have double immunofluorescence staining. CK7 and 16 in all ontogenetic period as well as Streptavidin-biotin immunoperoxidase staining medullary CK18 negative cells could be the candi- was performed as follows: thymic cryostat sections dates for ectodermaly derived epithelium. But the (5/m), which were fixed in acetone for 10 min, problem is how to explain the origins of other cell were first incubated with mAbs for a minimum of types. The simultaneous expression of various 60 min, washed in TBS, pH 7.6 for 10 min, followed common CK polypeptides during ontogeny favors, by incubation with secondary biotinylated antibodies although does not strongly prove, the hypothesis of (IgG subclass specific) diluted 1:100 in TBS for a common origin of thymic epithelium. Medullary 30 min. After washing in TBS, sections were incucells having common and constant CK polypeptides bated with streptavidin-peroxidase (1:100) for 8, 10, 18, and 19 in all ontogenetic periods could be 30 min. Revelation of the peroxidase activity was a common, maybe undifferentiated, TEC subset, performed by a 10-min incubation of the sections in Morphologically undifferentiated epithelial cells have 0.06% DAB (Serva, FRG) in 0.01% H202. Finally, been previously described in the corticomedullary slides were lightly counterstained with hematoxylin region and medulla (yon Gaudecher et al., 1986). and mounted in gelatin/glycerol medium. In conclusion, this work throws new light on the Double immunofluorescence was performed in development of thymic epithelium and raises many order to determine whether two different CK polyquestions about the functional significance of so peptides were present in the, same TEC. To prove extremely different CK polypeptide expressions in this, sections were incubated with mAbs mutually the thymus, differing in isotype specificity. The reaction was visualized using subclass-specific secondary reagents coupled with different dyes. The staining sequence MATERIALS AND METHODS was as follows: first, mAb, IgG subclass-specific FITC-coupled antibody; second, mAb .IgG subclass Animals specific biotinylated antibody, streptavidin-TR. Thymuses were obtained from A0 rats, bred at the Sometimes an opposite sequence of secondary antiFarm for Experimental Animals, Military" Medical bodies was applied. After washing following each Academy, Belgrade, of the following age groups: incubation, sections were mounted in glycerol and days 15, 17, and 19 of fetal life, day of birth, days 7 observed for green and red fluorescence under a and 14 or 6 weeks of postnatal life. Day 0 of preg- fluorescence microscope (Univar III). Specificity of nancy was determined by regular controls of the labeling was checked using appropriate controls, estral cycle and findings of spermatozoides in including single staining, omitting the first or vaginal smears. Samples were collected from 3-5 second mAb, as well as their replacement with an animals per time point, irrelevant mouse mAb of the same isotype (produced in our laboratory). All controls gave negative Antibodies and Reagents
results.
A panel of 10 mouse, anti-CK monoclonal antibodies
(Received August 20, 1989)
was used. Their specificity, isotype, dilution, and origin were given in Table 1. Secondary antibodies
(Accepted September 26, 1989)
ONTOGENY OF RAT THYMIC CYTOKERATINS
REFERENCES (oli4 M., Matanovi4 D., Hegadi Lj., and Duji4 A. (1988a). Heterogeneity of rat thymic epithelium by monoclonal antikeratin antibodies. Thymus 12: 123. (oli4 M., Matanovi4 D., Hegedi Lj., and Duji4 A. (1988b). Immunohistochemical characterization of rat thymic nonlymphoid cellsmI. Epithelial and mesenchymal components defined by monoclonal antibodies. Immunology 65: 277. (oli4. M., Jovanovi4 S., Mitrovi4 S., and Duji4 A. (1989). Immunohistochemical identification of six cytokeratin-defined subsets of the rat thymic epithelial cells. Thymus 13: 175. Cooper D., Schermer A., and Sun T.T. (1985). Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: Strategies, applications, and limitations. Lab. Invest. 52: 243. Cordier A.C., and Heremans J.E. (1975). Nude mouse embryo: Ectodermal nature of the primordial thymic defect. Scand. J. Immunol. 4: 193. Crouse D.A., Turpen J.B., and Sharp J.G. (1985). Thymic nonlymphoid cells. Surv. Immunol. Res. 4: 120. Gaudecker B. Von. (1986). The development of the human thymus microenvironments. In: The human thymus: Histophysiology and pathology, Muller-Hermelink H.K., Ed. (Berlin: Springer
Verlag), p. 1. Gelfand E.W., Dosch H.M., Shore A., Limbatibul S., and Lee J.W.W. (1980). Role of thymus in human T cells differentiation. In: Biological basis of immunodeficiency, Gelfand E.W., and Dosch H.M., Eds. (New York: Raven Press), p. 39. Haftek M., Staquet M.J., Viac J., Schmitt D., and Thivolet J. (1986). Immunogold labeling of keratin filaments in normal human epidermal cells with two anti-keratin monoclonal antibodies. J. Histochem. Cytochem. 34: 613. Kampinga J., Berger S., Boyd R., Brekelmans P., Coli4 M., Van Ewijk W., Kendall M., Ladyman H., Nieuwenhuis P., Ritter M., Schuurman H-J., and Tournefier A. (1989). Thymic epithelial antibodies: Immunohistological analysis and introduction of CTES nomenclature. Thymus 13: 165. Lampert I., and Ritter M.A. (1988). The origin of the diverse epithelial cells of the thymus: Is there a common stem cell? In: Thymus update, vol. 1, Kendall M.D., and Ritter M.A., eds. (London: Harwood Academic Publishers), p. 5. Lane E.B. (1982). Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J. Cell Biol. 92: 665. Laster A.J., Itoh T., Palker T.J., and Haynes B.F. (1986). The human thymic microenvironment: Thymic epithelium contain specific keratins associated with early and late stages of epidermal keratinocyte maturation. Differentiation. 31: 67.
75
Lazarides E. (1982). Intermediate filaments: A chemically heterogeneous, developmentally regulated class of proteins. Annu.. Rev. Biochem. 51: 219. Lobach D.F., and .Haynes B.F. (1987). Ontogeny of the human thymus during fetal development. J. Clin. Immunol. 7: 81. Mitrovi4 S., (oli4 M., and Popovi4 Lj. (in press). Comparative studies on the expression of SHC-9D8 antigen and cytokeratin 18 in rat epithelial cells. Period. Biol. Moll R., Franke W.W., and Schiller D.L. (1982). The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell 31: 11. Nicolas J.F., Reano A., Kaiserlian D., and Thivolet J. (1986). Epithelial cell heterogeneity in the guinea pig thymus: Immunohistochemical characterization of four thymic epithelial subsets defined by monoclonal antikeratin antibodies. Eur. J. Immunol. 16: 457. Nicolas J.F., Savino W., Reano A., Viac J., and Dardenne M. (1985). Heterogeneity of thymic epithelial cell (TEC) keratins: Immunohistochemical and biochemical evidence for a subset of highly differentiated TEC in the mouse. J. Histochem. Cytochem. 33: 687. Quinlan R.A., Schiller D.L., Hatzfeld M., Achstatter T., Moll R., Jorcano J.L., Magin T.M., and Franke W.W. (1985). Patterns of expression and organization of cytokeratin intermediate filaments. Annu. NY Acad. Sci. 455: 282. Ritter M.A., and Haynes B.F. (1987). Summary of thymic epithelium workshop. In: Leucocyte typing III. White cell differentiation antigens, McMichael, A., Beverley P., Hagg N., and Horton M. Eds. (Oxford: Oxford University Press), p. 247. Savino W., and Dardenne M. (1988a). Development studies on expression of monoclonal antibody-defined cytokeratins by thymic epithelial cells from normal and autoimmune mice. J. Histochem. Cytochem. 36: 1123. Savino W., and Dardenne M. (1988b). Immunohistochemical studies on a human thymic epithelial subset defined by the anti-cytokeratin 18 monoclonal antibody. Cell Tissue Res. 254: 255. Tolle H.G., Weber K., and Osborn M. (1985). Microinjection of monoclonal antibodies specific one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments. Eur. J. Cell Biol. 38: 234. Viac J., Reano A., Brochier J., Staquet M.J., Thivolet J. (1983). Reactivity pattern of monoclonal anti-keratin antibody.(KL 1). J. Invest. Dermatol. 81: 351. Willcox N., Shluep M., Ritter M.A., Schuurman H-J., NewsonDavis J., and Christensson B. (1987). Myasthenic and nonmyasthenic thymoma. An expansion of a minor cortical epithelial cell subset? Am. J. Pathol. 127: 447.
MEDIATORS of
INFLAMMATION
The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Gastroenterology Research and Practice Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Journal of
Hindawi Publishing Corporation http://www.hindawi.com
Diabetes Research Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
International Journal of
Journal of
Endocrinology
Immunology Research Hindawi Publishing Corporation http://www.hindawi.com
Disease Markers
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Volume 2014
Submit your manuscripts at http://www.hindawi.com BioMed Research International
PPAR Research Hindawi Publishing Corporation http://www.hindawi.com
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Volume 2014
Journal of
Obesity
Journal of
Ophthalmology Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Evidence-Based Complementary and Alternative Medicine
Stem Cells International Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Journal of
Oncology Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Parkinson’s Disease
Computational and Mathematical Methods in Medicine Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
AIDS
Behavioural Neurology Hindawi Publishing Corporation http://www.hindawi.com
Research and Treatment Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
Oxidative Medicine and Cellular Longevity Hindawi Publishing Corporation http://www.hindawi.com
Volume 2014
